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Objective We evaluated the effect of three-dimensional static and dynamic culture on the proliferation, distribution, and differentiation of rabbit mesenchymal stem cells (MSCs) in a porous scaffold via autograft for bone regeneration. Methods Rabbit MSCs were seeded in a porous hydroxyapatite scaffold (MSCs/scaffold), and then cultured in petri dishes and a bidirectional flow reactor for 4 weeks for osteogenetic induction in vitro . Metabolic assay of the culture medium was carried out every 2 days; glucose, lactic acid, and calcium concentrations in the medium were also examined. Cell distribution in the scaffold was examined histologically. Cultured MSCs/scaffolds were implanted in rabbit condyles for the evaluation of bone regeneration in vivo . Results Histological sections showed that cells cultured in petri dishes grew only around the scaffolds and seldom in the inner part. However, the scaffolds cultured with MSCs in a bioreactor were almost fully occupied by cells. Metabolic results revealed that the average concentration of glucose in the medium was decreased as cells propagated. Glucose consumption was observed in both static and dynamic cultures, but higher lactic acid production was found in the static culture. Calcium ion concentration was reduced significantly in the dynamic culture after the addition of an osteogenetic induction medium, indicating progression of mineralization. The in vivo results showed that about 80% of the defect in the condyles with dynamically cultured MSCs/scaffold implants was filled with new bone tissue, a proportion much higher than that in the petri dish-cultured MSCs/scaffolds, in which only half of the bone regeneration occurred in the cavity. Conclusion This study provides evidence of the effectiveness of dynamic in vitro MSC culture for robust osteogenesis and indicates that it may impart superior potential for bone regeneration in vivo .

Background Developing ultrasensitive and selective biological sensors has increased in interest for applications in disease detection, drug discovery, and biomedical diagnostics. This paper reports a CMOS-compatible technique for fabricating a silicon nanowire field-effect transistor. The nanowire sensor was applied to detect a fragment of the cancer-related interleukin-1β (IL-1β) gene, which is characterized as an indicator of breast, colon, lung, oral, head, and neck cancers. Methods We used the advanced integrated circuit (IC) technique to fabricate a back-gated nanowire field-effect transistor sensor. The dimensions of the nanowire were 60 nm in width and 20 μm in length. To enhance the sensitivity of the nanowire sensor, a plasma modification treatment with various parameters was employed on the surface of the device. Results The sensitivity of the silicon nanowire field-effect transistor could be improved after a 1-minute N2O plasma treatment, because the morphology of the detection region is rougher after the N2O plasma treatment. Hence, a more functional linker could be bound to the surface, thereby increasing the probability of DNA immobilization and hybridization. We used a specific sequence of the IL-1β gene to verify the sensitivity enhancement of the nanowire sensor by using plasma treatment. Conclusion The sensitivity and detection limit of the plasma-treated sensor can be extrapolated to 0.12/decade and 2.5fM, respectively. The detection results demonstrate that real-time monitoring of the expression of IL-1β using nanowire sensors could be useful for evaluating cancer treatment.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

Previous studies have shown that collagen gel overlay induced selective proteolysis of focal adhesion complex proteins in Madin-Darby canine kidney (MDCK) cells. In this study, we examined whether morphological and biochemical changes were present in cells cultured on collagen gel. We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas, but not vinculin, were decreased within 1 h when MDCK cells were cultured on collagen gel. Collagen gel-induced selective decrease of focal adhesion proteins was observed in all lines of cells examined, including epithelial, fibroblastic, and cancer cells. Matrigel also induced selective down-regulation of focal adhesion proteins. However, cells cultured on collagen gel- or matrigel-coated dishes did not show any changes of focal adhesion proteins. These data suggest that the physical nature of the gel, i.e. the rigidity, is involved in the expression of focal adhesion proteins. The collagen gel-induced down-regulation of focal adhesion complex proteins was caused by reduction of protein synthesis and activation of proteases such as calpain. Overexpression of a dominant negative mutant of discoidin domain receptor 1 (DDR1) or FAK-related non-kinase (FRNK) did not prevent collagen gel-induced down-regulation of the focal adhesion complex protein, whereas an anti-alpha2beta1 integrin-neutralizing antibody completely blocked it. Taken together, our results indicate that the rigidity of collagen gel controls the expression of focal adhesion complex proteins, which is mediated by alpha2beta1 integrin but not DDR1.

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRα1. In the developing kidney, RET, GDNF, and GFRα1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.

Learning controllers are usually subordinate to conventional controllers in governing multiple-joint robot motion, in spite of their ability to generalize, because learning space complexity and motion variety require them to consume excessive amount of memory when they are employed as major roles in motion governing. We propose using a fuzzy neural network (FNN) to learn and analyze robot motions so that they can be classified according to similarity. After classification, the learning controller can then be designed to govern robot motions according to their similarities without consuming excessive memory resources.

Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding proteins belonging to the calmodulin superfamily. Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca2+-binding loops were replaced by reporter Trp residues. Using fluorescence spectroscopy and biochemical assays, we found that binding of Ca2+ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energyE a = 9.3 kcal/mol. When Tyr99adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys99 is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurements indicated that GCAP1 rapidly loses its bound Ca2+ (k −1 = 72 s−1 at 37 °C) and was estimated to associate with Ca2+ at a rate (k 1 > 2 × 108 m −1 s−1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response.

Attempts were made to examine the effect of growth inhibitors on regulation of Na,K-ATPase in primary cultures of renal proximal tubule cells. We observed that both TGF-beta and butyrate induced dose dependent decrease of Na,K-ATPase activity. The time course studies showed that the effect of TGF-beta preceded the effect of butyrate on inhibition of Na,K-ATPase activity. Both butyrate- and TGF-beta-induced inhibition of this enzyme were in general mediated by transcriptional decreases of alpha and beta mRNA and therefore also protein abundance. Like its effect on Na,K-ATPase activity, TGF-beta compared to butyrate induced much earlier decline in Na,K-ATPase alpha and beta mRNA and protein abundance. Butyrate did not affect gene expression of TGF-beta and anti-TGF-beta antibody had no effect on butyrate-induced inhibition of DNA synthesis. Obviously, both butyrate and TGF-beta can inhibit the expression of Na,K-ATPase alpha and beta mRNA, but TGF-beta does not mediate butyrate's effect.

Upon excitation by 280 nm, the intensity decay of the 2 tyrosine residues (residues 10 and 109) of rabbit skeletal muscle troponin C is resolved into three components. The anisotropy decay in the absence of divalent cation is biphasic with a short correlation time of 0.67 ns and a long correlation time of 9.23 ns. The limiting anisotropy is 0.225, considerably lower than the value expected for immobilized tyrosine. Upon excitation by 290 nm, the anisotropy decay is also biphasic, and the limiting anisotropy increases to 0.274. The recovery of anisotropy by excitation at a wave-length near the red edge of the tyrosine absorption spectrum is evidence of fluorescence resonance energy transfer between the two tyrosines. For energy transfer to occur, the average separation between the 2 tyrosines is unlikely much larger than the Forster distance Ro, congruent to 10 A, and this close proximity of the residues would require a highly distorted dumbbell shape of troponin C in solution. These results are consistent with a flexible central helix, which either has a segmental flexibility with large amplitude or results in a spectrum of conformations including those in which the two globular domains are in a very close proximity.

Op18 is a widely expressed, cell cycle-regulated, phosphoprotein involved in signal transduction of a variety of stimuli. In actively proliferating Jurkat T cells which express Op18 at high level, phorbol 12-myristate 13-acetate (PMA) treatment induces a rapid increase in the level of several Op18 phosphorylated forms. To determine phosphorylation sites involved in the PMA effect, the major Op18 phosphorylated forms were resolved in Jurkat T cells, before and after treatment with PMA, using preparative immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. Tryptic fragments of phosphorylated Op18 were analyzed by two-dimensional thin layer peptide mapping and were resolved by reverse-phase high performance liquid chromatography prior to analysis by matrix-assisted laser desorption ionization mass spectrometry. Phosphorylation sites were identified by further treatment of the proteolytic fragments with different enzymes and determination of the mass shifts by matrix-assisted laser desorption ionization mass spectrometry. Two major phosphorylation sites were identified. Low constitutive levels of phosphorylation at Ser25 and Ser38 in Op18a and Op18b was demonstrated. Treatment with PMA resulted in enhanced phosphorylation of Ser25 in Op18a and of both Ser25 and Ser38 in Op18b. Taken together with prior studies of Op18 phosphorylation, the data suggest that Op18 phosphorylation occurs at identical sites in different tissues and organisms.

We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility.

We have determined six molecular distances among four sites in the binary complex formed between troponin C (TnC) and troponin I (TnI) by fluorescence resonance energy transfer between donor and acceptor probes that were either an intrinsic fluorophore (Trp158 of TnI) or extrinsic probes attached to the sites. The three extrinsic probes were dansylaziridine (DNZ), N'-(iodoacetyl)-N'-(8-sulfo-1-naphthyl)ethylenediamine (IAEDANS) and 5-(iodoacetamido)eosin (IAE). The four fluorophores provided four donor-acceptor pairs: DNZ----IAE, Trp----IAEDANS, IAEDANS----IAE, and Trp----DNZ. They allowed determinations of separations between specific sites from measurements of energy transfer from (1) Met25 (DNZ) to Cys98 (IAE) in TnC, (2) Trp158 to Cys133 (IAEDANS) in TnI, (3) Cys98 (IAEDANS) of TnC to Cys133(IAE) of TnI, (4) Trp158 of TnI to Cys98(IAEDANS) of TnC, and (6) Met25(DNZ) of TnC to Cys133(IAE) of TnI. Distance (1) in TnC was little affected when the isolated protein was complexed with TnI, whereas distance (2) in TnI increased by 6A (29%) when TnI was incorporated into the binary complex. In the presence of EGTA, the six donor-acceptor separations (R) in the complex were in the range 28 to 57 A based on kappa 2 = 2/3. Mg2+ had only small effects on R, but Ca2+ induced substantial increases or decreases of R in five of the six distances. These changes were not accompanied by significant changes in the axial depolarization of the fluorophores. The results indicate global structural perturbations of regions of the two proteins in the complex by Ca2+ binding to the TnC, and suggest that large-scale movements of domains of troponin subunits may be the initial molecular events that occur in the transmission of the Ca2+ signal in the regulation of contraction by calcium.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587–593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.